Parkinson's Disease (PD) is a major health burden on the ageing population in the United States and elsewhere. The identification of gene defects that cause the disease in rare families may lead to new insights into the pathophysiology of the more common sporadic forms. One particular gene linked to PD that is especially relevant to the sporadic disease is alpha-synuclein (ASYN), as its protein product is the major component of Lewy Bodies. Furthermore, multiplication of the ASYN locus is sufficient to cause PD in humans. Therefore, elucidating the normal and aberrant functions of ASYN, as well as the manner of its regulation, may lead to a better understanding of the pathophsyiology of PD and the development of novel therapies. We have previously found that mutant ASYN can impair protein degradation through the lysosomes and the ubiquitin-proteasome system. Defects of protein handling may also occur in other genetic or sporadic forms of PD, and may form a common pathophysiological basis for this disorder. We have proposed that the impact on the lysosomes may be mediated by an inhibition by mutant ASYN of the pathway of Chaperone-Mediated Autophagy (CMA), which normally degrades ASYN. However, these results were largely achieved in an artificial setting, and the degradation of ASYN and its impact on proteolytic systems is controversial. We propose to first establish that ASYN is degraded by CMA in neuronal cells, by manipulating Lamp2a, the receptor for CMA and its rate-limiting step, and by examining the degradation of ASYN when it is not targeted to the CMA pathway. Mutant ASYNs that do not affect CMA will also be examined for their relative toxic effects, to ascertain whether ASYN toxicity is mediated in part by CMA blockade. We have recently found that ASYN can be secreted and exert toxic effects, and we will examine whether such toxicity, which may be physiologically relevant, is related to the inhibition of UPS and/or lysosomal function in neuronal cells. Lastly, we will examine whether the effects of ASYN on protein degradation systems in neuronal cell lines are applicable in primary neuronal cell cultures. These studies will help to decipher the post-translational regulation of ASYN levels, which is critical for its steady-state levels and its impact on neuronal physiology, and its effects on protein degradation pathways, which may be critical for its pathogenic effects. [unreadable] [unreadable] [unreadable]